The present invention relates generally to polyketides and genes for their synthesis. In particular, the present invention relates to the isolation and characterization of novel polyketide synthase and nonribosomal peptide synthetase genes from Sorangium cellulosum that are necessary for the biosynthesis of epothilones A and B.
Polyketides are compounds synthesized from two-carbon building blocks, the xcex2-carbon of which always carries a keto group, thus the name polyketide. These compounds include many important antibiotics, immunosuppressants, cancer chemotherapeutic agents, and other compounds possessing a broad range of biological properties. The tremendous structural diversity derives from the different lengths of the polyketide chain, the different side-chains introduced (either as part of the two-carbon building blocks or after the polyketide backbone is formed), and the stereochemistry of such groups. The keto groups may also be reduced to hydroxyls, enoyls, or removed altogether. Each round of two-carbon addition is carried out by a complex of enzymes called the polyketide synthase (PKS) in a manner similar to fatty acid biosynthesis.
The biosynthetic genes for an increasing number of polyketides have been isolated and sequenced. For example, see U.S. Pat. Nos. 5,639,949, 5,693,774, and 5,716,849, all of which are incorporated herein by reference, which describe genes for the biosynthesis of soraphen. See also, Schupp et al., FEMS Microbiology Letters 159: 201-207 (1998) and WO 98/07868, which describe genes for the biosynthesis of rifamycin, and U.S. Pat. No. 5,876,991, which describes genes for the biosynthesis of tylactone, all of which are incorporated herein by reference. The encoded proteins generally fall into two types: type I and type II. Type I proteins are polyfunctional, with several catalytic domains carrying out different enzymatic steps covalently linked together (e.g. PKS for erythromycin, soraphen, rifamycin, and avermectin (MacNeil et al., in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al.), American Society for Microbiology, Washington D. C. pp. 245-256 (1993)); whereas type 11 proteins are monofunctional (Hutchinson et al., in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al), American Society for Microbiology, Washington D. C. pp. 203-216 (1993)).
For the simpler polyketides such as actinorhodin (produced by Streptomyces coelicolor), the several rounds of two-carbon additions are carried out iteratively on PKS enzymes encoded by one set of PKS genes. In contrast, synthesis of the more complicated compounds such as erythromycin and soraphen involves PKS enzymes that are organized into modules, whereby each module carries out one round of two-carbon addition (for review, see Hopwood et al, in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al.), American Society for Microbiology, Washington D. C., pp. 267-275 (1993)).
Complex polyketides and secondary metabolites in general may contain substructures that are derived from amino acids instead of simple carboxylic acids. Incorporations of these building blocks are accomplished by non-ribosomal polypeptide synthetases (NRPSs). NRPSs are multienzymes that are organized in modules. Each module is responsible for the addition (and the additional processing, if required) of one amino acid building block. NRPSs activate amino acids by forming aminoacyl-adenylates, and capture the activated amino acids on thiol groups of phophopantheteinyl prosthetic groups on peptidyl carrier protein domains. Further, NRPSs modify the amino acids by epimerization, N-methylation, or cyclization if necessary, and catalyse the formation of peptide bonds between the enzyme-bound amino acids. NRPSs are responsible for the biosynthesis of peptide secondary metabolites like cyclosporin, could provide polyketide chain terminator units as in rapamycin, or form mixed systems with PKSs as in yersiniabactin biosynthesis.
Epothilones A and B are 16-membered macrocyclic polyketides with an acylcysteine-derived starter unit that are produced by the bacterium Sorangium cellulosum strain So ce90(Gerth et al., J. Antibiotics 49: 560-563 (1996), incorporated herein by reference). The structure of epothilone A and B wherein R signifies hydrogen (epothilone A) or methyl (epothilone B) is: 
The epothilones have a narrow antifungal spectrum and especially show a high cytotoxicity in animal cell cultures (see, Hxc3x6fleet al., Patent DE 4138042 (1993), incorporated herein by reference). Of significant importance, epothilones mimic the biological effects of taxol, both in vivo and in cultured cells (Bollag et al., Cancer Research 55: 2325-2333 (1995), incorporated herein by reference). Taxol and taxotere, which stabilize cellular microtubules, are cancer chemotherapeutic agents with significant activity against various human solid tumors (Rowinsky et al., J. Natl. Cancer Inst. 83: 1778-1781 (1991)). Competition studies have revealed that epothilones act as competitive inhibitors of taxol binding to microtubules, consistent with the interpretation that they share the same microtubule-binding site and possess a similar microtubule affinity as taxol. However, epothilones enjoy a significant advantage over taxol in that epothilones exhibit a much lower drop in potency compared to taxol against a multiple drug-resistant cell line (Bollag et al. (1995)). Furthermore, epothilones are considerably less efficiently exported from the cells by P-glycoprotein than is taxol (Gerth et al. (1996)). In addition, several epothilone analogs have been synthesized that have a superior cytotoxic activity as compared to epothilone A or epothilone B as demonstrated by their enhanced ability to induce the polymerization and stabilization of microtubules (WO 98/25929, incorporated herein by reference).
Despite the promise shown by the epothilones as anticancer agents, problems pertaining to the production of these compounds presently limit their commercial potential. The compounds are too complex for industrial-scale chemical synthesis and so must be produced by fermentation. Techniques for the genetic manipulation of myxobacteria such as Sorangium cellulosum are described in U.S. Pat. No.5,686,295, incorporated herein by reference. However, Sorangium cellulosum is notoriously difficult to ferment and production levels of epothilones are therefore low. Recombinant production of epothilones in heterologous hosts that are more amenable to fermentation could solve current production problems. However, the genes that encode the polypeptides responsible for epothilone biosynthesis have heretofore not been isolated. Furthermore, the strain that produces epothilones, i.e. So ce90, also produces at least one additional polyketide, spirangien, which would be expected to greatly complicate the isolation of the genes particularly responsible for epothilone biosynthesis.
Therefore, in view of the foregoing, one object of the present invention is to isolate the genes that are involved in the synthesis of epothilones, particularly the genes that are involved in the synthesis of epothilones A and B in myxobacteria of the Sorangium/-Polyangium group, i.e., Sorangium cellulosum strain So ce90. A further object of the invention is to provide a method for the recombinant production of epothilones for application in anticancer formulations.
In furtherance of the aforementioned and other objects, the present invention unexpectedly overcomes the difficulties set forth above to provide for the first time a nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of epothilone. In a preferred embodiment, the nucleotide sequence is isolated from a species belonging to Myxobacteria, most preferably Sorangium cellulosum. 
In another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: SEQ ID NO:2, amino acids 11-437 of SEQ ID NO:2, amino acids 543-864 of SEQ ID NO:2, amino acids 974-1273 of SEQ ID NO:2, amino acids 1314-1385 of SEQ ID NO:2, SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, amino acids 1344-1351 of SEQ ID NO:3, SEQ ID NO:4, amino acids 7-432 of SEQ ID NO:4, amino acids 539-859 of SEQID NO:4, amino acids 869-1037 of SEQ ID NO:4, amino acids 1439-1684 of SEQ ID NO:4, amino acids 1722-1792 of SEQ ID NO:4, SEQ ID NO:5, amino acids 39-457 of SEQ ID NO:5, amino acids 563-884 of SEQ ID NO:5, amino acids 1147-1399 of SEQ ID NO:5, amino acids 1434-1506 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 3886-4048 of SEQ ID NO:5, amino acids 4433-4719 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, SEQ ID NO:6, amino acids 35-454 of SEQ ID NO:6, amino acids 561-881 of SEQ ID NO:6, amino acids 1143-1393 of SEQ ID NO:6, amino acids 1430-1503 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, amino acids 2053-2373 of SEQ ID NO:6, amino acids 2383-2551 of SEQ ID NO:6, amino acids 2671-3045 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, SEQ ID NO:7, amino acids 32-450 of SEQ ID NO:7, amino acids 556-877 of SEQ ID NO:7, amino acids 887-1051 of SEQ ID NO:7, amino acids 1478-1790 of SEQ ID NO:7, amino acids 1810-2055 of SEQ ID NO:7, amino acids 2093-2164 of SEQ ID NO:7, amino acids 2165-2439 of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:22.
In a more preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, amino acids 11-437 of SEQ ID NO:2, amino acids 543-864 of SEQ ID NO:2, amino acids 974-1273 of SEQ ID NO:2, amino acids 1314-1385 of SEQ ID NO:2, SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, amino acids 1344-1351 of SEQ ID NO:3, SEQ ID NO:4, amino acids 7-432 of SEQ ID NO:4, amino acids 539-859 of SEQ ID NO:4, amino acids 869-1037 of SEQ ID NO:4, amino acids 1439-1684 of SEQ ID NO:4, amino acids 1722-1792 of SEQ ID NO:4, SEQ ID NO:5, amino acids 39-457 of SEQ ID NO:5, amino acids 563-884 of SEQ ID NO:5, amino acids 1147-1399 of SEQ ID NO:5, amino acids 1434-1506 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 3886-4048 of SEQ ID NO:5, amino acids 4433-4719 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, SEQ ID NO:6, amino acids 35-454 of SEQ ID NO:6, amino acids 561-881 of SEQ ID NO:6, amino acids 1143-1393 of SEQ ID NO:6, amino acids 1430-1503 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, amino acids 2053-2373 of SEQ ID NO:6, amino acids 2383-2551 of SEQ ID NO:6, amino acids 2671-3045 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, SEQ ID NO:7, amino acids 32-450 of SEQ; ID NO:7, amino acids 556-877 of SEQ ID NO:7, amino acids 887-1051 of SEQ ID NO:7, amino acids 1478-1790 of SEQ ID NO:7, amino acids 1810-2055 of SEQ ID NO:7, amino acids 2093-2164 of SEQ ID NO:7, amino acids 2165-2439 of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:22.
In yet another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence is substantially similar to a nucleotide sequence selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucleotides,10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
In an especially preferred embodiment, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence is selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucieotides 10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
In yet another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being stably transformed into a host cell. Still further, the present invention provides a recombinant host cell comprising such a chimeric gene, wherein the host cell is capable of expressing the nucleotide sequence that encodes at least one polypeptide necessary for the biosynthesis of an epothilone. In a preferred embodiment, the recombinant host cell is a bacterium belonging to the order Actinomycet ales, and in a more preferred embodiment the recombinant host cell is a strain of Streptomyces. In other embodiments, the recombinant host cell is any other bacterium amenable to fermentation, such as a pseudomonad or E. coli. Even further, the present invention provides a Bac clone comprising a nucleic acid molecule of the invention, preferably Bac clone pEPO15.
In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an epothilone synthase domain.
According to one embodiment, the epothilone synthase domain is a xcex2-ketoacyl-synthase (KS) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7. According to this embodiment, said KS domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is an acyltransferase (AT) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. According to this embodiment, said AT domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1.
According to still another embodiment, the epothilone synthase domain is an enoyl reductase (ER) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7. According to this embodiment, said ER domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID N6:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is an acyl carrier protein (ACP) domain, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. According to this embodiment, said ACP domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is a dehydratase (DH) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. According to this embodiment, said DH domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1.
According to yet another embodiment, the epothilone synthase domain is a xcex2-ketoreductase (KR) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. According to this embodiment, said KR domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1.
According to an additional embodiment, the epothilone synthase domain is a methyltransferase (MT) domain comprising an amino acid sequence substantially similar to amino acids 2671-3045 of SEQ ID NO:6. According to this embodiment, said MT domain preferably comprises amino acids 2671-3045 of SEQ ID NO:6. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to nucleotides 51534-52657 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of nucleotides 51534-52657 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is nucleotides 51534-52657 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is a thioesterase (TE) domain comprising an amino acid sequence substantially similar to amino acids 2165-2439 of SEQ ID NO:7. According to this embodiment, said TE domain preferably comprises amino acids 2165-2439 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to nucleotides 61427-62254 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of nucleotides 61427-62254 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is nucleotides 61427-62254 of SEQ ID NO:1.
In still another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a non-ribosomal peptide synthetase, wherein said non-ribosomal peptide synthetase comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, and amino acids 1344-1351 of SEQ ID NO:3. According to this embodiment, said non-ribosomal peptide synthetase preferably comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, and amino acids 1344-1351 of SEQ ID NO:3. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1.
The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2-23.
In accordance with another aspect, the present invention also provides methods for the recombinant production of polyketides such as epothilones in quantities large enough to enable their purification and use in pharmaceutical formulations such as those for the treatment of cancer. A specific advantage of these production methods is the chirality of the molecules produced; production in transgenic organisms avoids the generation of populations of racemic mixtures, within which some enantiomers may have reduced activity. In particular, the present invention provides a method for heterologous expression of epothilone in a recombinant host, comprising: (a) introducing into a host a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention that comprises a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of epothilone; and (b) growing the host in conditions that allow biosynthesis of epothilone in the host. The present invention also provides a method for producing epothilone, comprising: (a) expressing epothilone in a recombinant host by the aforementioned method; and (b) extracting epothilone from the recombinant host.
According to still another aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence that consists of an epothilone synthase domain.
According to one embodiment, the epothilone synthase domain is a xcex2-ketoacyl-synthase (KS) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7. According to this embodiment, said KS domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7;
According to another embodiment, the epothilone synthase domain is an acyltransferase (AT) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. According to this embodiment, said AT domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7.
According to still another embodiment, the epothilone synthase domain is an enoyl reductase (ER) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7. According to this embodiment, said ER domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7.
According to another embodiment, the epothilone synthase domain is an acyl carrier protein (ACP) domain, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. According to this embodiment, said ACP domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7.
According to another embodiment, the epothilone synthase domain is a dehydratase (DH) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. According to this embodiment, said DH domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7.
According to yet another embodiment, the epothilone synthase domain is a xcex2-ketoreductase (KR) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. According to this embodiment, said KR domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7.
According to an additional embodiment, the epothilone synthase domain is a methyltransferase (MT) domain comprising an amino acid sequence substantially similar to amino acids 2671-3045 of SEQ ID NO:6. According to this embodiment, said MT domain preferably comprises amino acids 2671-3045 of SEQ ID NO:6.
According to another embodiment, the epothilone synthase domain is a thioesterase (TE) domain comprising an amino acid sequence substantially similar to amino acids 2165-2439 of SEQ ID NO:7. According to this embodiment, said TE domain preferably comprises amino acids 2165-2439 of SEQ ID NO:7.
Other aspects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
Associated With/Operatively Linked: Refers to two DNA sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be xe2x80x9cassociated withxe2x80x9d a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
Chimeric Gene: A recombinant DNA sequence in which a promoter or regulatory DNA sequence is operatively linked to, or associated with, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence. The regulator DNA sequence of the chimeric gene is not normally operatively linked to the associated DNA sequence as found in nature.
Coding DNA Sequence: A DNA sequence that is translated in an organism to produce a protein.
Domain: That part of a polyketide synthase necessary for a given distinct activity. Examples include acyl carrier protein (ACP), xcex2-ketosynthase (KS), acyltransferase (AT), xcex2-ketoreductase (KR), dehydratase (DH), enoylreductase (ER), and thioesterase (TE) domains.
Epothilones: 16-membered macrocyclic polyketides naturally produced by the bacterium Sorangium cellulosum strain So ce90, which mimic the biological effects of taxol. In this application, xe2x80x9cepothilonexe2x80x9d refers to the class of polyketides that includes epothilone A and epothilone B, as well as analogs thereof such as those described in WO 98/25929.
Epothilone Synthase: A polyketide synthase responsible for the biosynthesis of epothilone.
Gene: A defined region that is located within a genome and that, besides the aforementioned coding DNA sequence, comprises other, primarily regulatory, DNA sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
Heterologous DNA Sequence: A DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
Homologous DNA Sequence: A DNA sequence naturally associated with a host cell into which it is introduced.
Homologous Recombination: Reciprocal exchange of DNA fragments between homologous DNA molecules.
Isolated: In the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
Module: A genetic element encoding all of the distinct activities required in a single round of polyketide biosynthesis, i.e., one condensation step and all the xcex2-carbonyl processing steps associated therewith. Each module encodes an ACP, a KS, and an AT activity to accomplish the condensation portion of the biosynthesis, and selected postcondensation activities to effect the xcex2-carbonyl processing.
NRPS: A non-ribosomal polypeptide synthetase, which is a complex of enzymatic activities responsible for the incorporation of amino acids into secondary metabolites including, for example, amino acid adenylation, epimerization, N-methylation, cyclization, peptidyl carrier protein, and condensation domains. A functional NRPS is one that catalyzes the incorporation of an amino acid into a secondary metabolite.
NRPS gene: One or more genes encoding NRPSs for producing functional secondary metabolites, e.g., epothilones A and B, when under the direction of one or more compatible control elements.
Nucleic Acid Molecule: A linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.
ORF: Open Reading Frame.
PKS: A polyketide synthase, which is a complex of enzymatic activities (domains) responsible for the biosynthesis of polyketides including, for example, ketoreductase, dehydratase, acyl carrier protein, enoylreductase, ketoacyl ACP synthase, and acyltransferase. A functional PKS is one that catalyzes the synthesis of a polyketide.
PKS Genes: One or more genes encoding various polypeptides required for producing functional polyketides, e.g., epothilones A and B, when under the direction of one or more compatible control elements.
Substantially Similar: With respect to nucleic acids, a nucleic acid molecule that has at least 60 percent sequence identity with a reference nucleic acid molecule. In a preferred embodiment, a substantially similar DNA sequence is at least 80% identical to a reference DNA sequence; in a more preferred embodiment, a substantially similar DNA sequence is at least 90% identical to a reference DNA sequence; and in a most preferred embodiment, a substantially similar DNA sequence is at least 95% identical to a reference DNA sequence. A substantially similar DNA sequence preferably encodes a protein or peptide having substantially the same activity as the protein or peptide encoded by the reference DNA sequence. A substantially similar nucleotide sequence typically hybridizes to a reference nucleic acid molecule, or fragments thereof, under the following conditions: hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 50xc2x0 C.; wash with 2xc3x97SSC, 1% SDS, at 50xc2x0 C. With respect to proteins or peptides, a substantially similar amino acid sequence is an amino acid sequence that is at least 90% identical to the amino acid sequence of a reference protein or peptide and has substantially the same activity as the reference protein or peptide.
Transformation: A process for introducing heterologous nucleic acid into a host cell or organism.
Transformed/Transgenic/Recombinant: Refers to a host organism such as a bacterium into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A xe2x80x9cnon-transformedxe2x80x9d, xe2x80x9cnon-transgenicxe2x80x9d, or xe2x80x9cnon-recombinantxe2x80x9d host refers to a wild-type organism, i.e., a bacterium, which does not contain the heterologous nucleic acid molecule.
Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore, (Xaa; X) represents any amino acid.
SEQ ID NO:1 is the nucleotide sequence of a 68750 bp contig containing 22 open reading frames (ORFs), which comprises the epothilone biosynthesis genes. SEQ ID NO:2 is the protein sequence of a type I polyketide synthase (EPOS A) encoded by epoA (nucleotides 7610-11875 of SEQ ID NO:1). SEQ ID NO:3 is the protein sequence of a non-ribosomal peptide synthetase (EPOS P) encoded by epoP (nucleotides 11872-16104 of SEQ ID NO:1).
SEQ ID NO:4 is the protein sequence of a type I polyketide synthase (EPOS B) encoded by epoB (nucleotides 16251-21749 of SEQ ID NO:1).
SEQ ID NO:5 is the protein sequence of a type I polyketide synthase (EPOS C) encoded by epoC (nucleotides 21746-43519 of SEQ ID NO:1).
SEQ ID NO:6 is the protein sequence of a type I polyketide synthase (EPOS D) encoded by epoD (nucleotides 43524-54920 of SEQ ID NO:1).
SEQ ID NO:7 is the protein sequence of a type I polyketide synthase (EPOS E) encoded by epoE (nucleotides 54935-62254 of SEQ ID NO:1).
SEQ ID NO:8 is the protein sequence of a cytochrome P450 oxygenase homologue (EPOS F) encoded by epoF (nucleotides 62369-63628 of SEQ ID NO:1).
SEQ ID NO:9 is a partial protein sequence (partial Orf 1) encoded by orfl (nucleotides 1-1826 of SEQ ID NO:1).
SEQ ID NO:10 is a protein sequence (Orf 2) encoded by orf2 (nucleotides 3171-1900 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:11 is a protein sequence (Orf 3) encoded by orf3 (nucleotides 3415-5556; of SEQ ID NO:1).
SEQ ID NO:12 is a protein sequence (Orf 4) encoded by orf4 (nucleotides 5992-5612 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:13 is a protein sequence (Orf 5) encoded by orf5 (nucleotides 6226-6675 of SEQ ID NO:1). SEQ ID NO:14 is a protein sequence (Orf 6) encoded by orf6 (nucleotides 63779-64333 of SEQ IDNO:1). SEQ ID NO:15 is a protein sequence (Orf 7) encoded by orf7 (nucleotides 64290-63853 on the reverse complement strand of SEQ ID NO:1). SEQ ID NO:16 is a protein sequence (Orf 8) encoded by orf8 (nucleotides 64363-64920 of SEQ ID NO:1). SEQ ID NO:17 is a protein sequence (Orf 9) encoded by orf9 (nucleotides 64727-64287 on the reverse complement strand of SEQ ID NO:1). SEQ ID NO:18 is a protein sequence (Orf 10) encoded by orf10 (nucleotides 65063-65767 of SEQ ID NO:1). SEQ ID NO:19 is a protein sequence (Orf 11) encoded by orf 11 (nucleotides 65874-65008 on the reverse complement strand of SEQ ID NO:1). SEQ ID NO:20 is a protein sequence (Orf 12) encoded by orf12 (nucleotides 66338-65871 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:21 is a protein sequence (Orf 13) encoded by orfl3 (nucleotides 66667-67137 of SEQ ID NO:1).
SEQ ID NO:22 is a protein sequence (Orf 14) encoded by orf14 (nucleotides 67334-68251 of SEQ ID NO:1).
SEQ ID NO:23 is a partial protein sequence (partial Orf 15) encoded by orf5 (nucleotides 68346-68750 of SEQ ID NO:1).
SEQ ID NO:24 is the universal reverse PCR primer sequence.
SEQ ID NO:25 is the universal forward PCR primer sequence.
SEQ ID NO:26 is the NH24 end xe2x80x9cBxe2x80x9d PCR primer sequence.
SEQ ID NO:27 is the NH2 end xe2x80x9cAxe2x80x9d PCR primer sequence.
SEQ ID NO:28 is the NH2 end xe2x80x9cBxe2x80x9d PCR primer sequence.
SEQ ID NO:29 is the pEPO15-NH6 end xe2x80x9cBxe2x80x9d PCR primer sequence.
SEQ ID NO:30 is the pEPO15-H2.7 end xe2x80x9cAxe2x80x9d PCR primer sequence.
The following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent.
The genes involved in the biosynthesis of epothilones can be isolated using the techniques according to the present invention. The preferable procedure for the isolation of epothilone biosynthesis genes requires the isolation of genomic DNA from an organism identified as producing epothilones A and B, and the transfer of the isolated DNA on a suitable plasmid or vector to a host organism that does not normally produce the polyketide, followed by the identification of transformed host colonies to which the epothilone-producing ability has been conferred. Using a technique such as xcex::Tn5 transposon mutagenesis (de Bruijn and Lupski, Gene 27: 131-149 (1984)), the exact region of the transforming epothilone-conferring DNA can be more precisely defined. Alternatively or additionally, the transforming epothilone-conferring DNA can be cleaved into smaller fragments and the smallest that maintains the epothilone-conferring ability further characterized. Whereas the host organism lacking the ability to produce epothilone may be a different species from the organism from which the polyketide derives, a variation of this technique involves the transformation of host DNA into the same host that has had its epothilone-producing ability disrupted by mutagenesis. In this method, an epothilone-producing organism is mutated and nonepothilone-producing mutants are isolated. These are then complemented by genomic DNA isolated from the epothilone-producing parent strain.
A further example of a technique that can be used to isolate genes required for epothilone biosynthesis is the use of transposon mutagenesis to generate mutants of an epothilone-producing organism that, after mutagenesis, fails to produce the polyketide. Thus, the region of the host genome responsible for epothilone production is tagged by the transposon and can be recovered and used as a probe to isolate the native genes from the parent strain. PKS genes that are required for the synthesis of polyketides and that are similar to known PKS genes may be isolated by virtue of their sequence homology to the biosynthetic genes for which the sequence is known, such as those for the biosynthesis of rifamycin or soraphen. Techniques suitable for isolation by homology include standard library screening by DNA hybridization.
Preferred for use as a probe molecule is a DNA fragment that is obtainable from a gene or another DNA sequence that plays a part in the synthesis of a known polyketide. A preferred probe molecule comprises a 1.2 kb SmaI DNA fragment encoding the ketosynthase domain of the fourth module of the soraphen PKS (U.S. Pat. No. 5,716,849), and a more preferred probe molecule comprises the xcex2-ketoacyl synthase domains from the first and second modules of the rifamycin PKS (Schupp et al., FEMS Microbiology Letters 159: 201-207 (1998)). These can be used to probe a gene library of an epothilone-producing microorganism to isolate the PKS genes responsible for epothilone biosynthesis.
Despite the well-known difficulties with PKS gene isolation in general and despite the difficulties expected to be encountered with the isolation of epothilone biosynthesis genes in particular, by using the methods described in the instant specification, biosynthetic genes for epothilones A and B can surprisingly be cloned from a microorganism that produces that polyketide. Using the methods of gene manipulation and recombinant production described in this specification, the cloned PKS genes can be modified and expressed in transgenic host organisms.
The isolated epothilone biosynthetic genes can be expressed in heterologous hosts to enable the production of the polyketide with greater efficiency than might be possible from native hosts. Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, heterologous genes can be expressed in Streptomyces and other actinomycetes using techniques such as those described in McDaniel et al, Science 262: 1546-1550 (1993) and Kao et al., Science 265: 509-512 (1994), both of which are incorporated herein by reference. See also, Rowe et al., Gene 216: 215-223 (1998); Holmes et al., EMBO Joumal 12(8): 3183-3191 (1993) and Bibb et al., Gene 38: 215-226 (1985), all of which are incorporated herein by reference.
Alternately, genes responsible for polyketide biosynthesis, i.e., epothilone biosynthetic genes, can also be expressed in other host organisms such as pseudomonads and E. coli. Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, PKS genes have been sucessfully expressed in E. coli using the pT7-7 vector, which uses the T7 promoter. See, Tabor et al., Proc. Natl. Acad. Sci. USA 82: 1074-1078 (1985), incorporated herein by reference. In addition, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli, either in transcriptional or translational fusion, behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector such as pKK223-3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al, American Society for Microbiology, Washington (1993)).
Other expression systems that may be used with the epothilone biosynthetic genes of the invention include yeast and baculovirus expression systems. See, for example, xe2x80x9cThe Expression of Recombinant Proteins in Yeasts,xe2x80x9d Sudbery, P. E., Curr. Opin. Biotechnol. 7(5): 517-524 (1996); xe2x80x9cMethods for Expressing Recombinant Proteins in Yeast,xe2x80x9d Mackay, et al., Editor(s): Carey, Paul R., Protein Eng. Des. 105-153, Publisher: Academic, San Diego, Calif. (1996); xe2x80x9cExpression of heterologous gene products in yeast,xe2x80x9d Pichuantes, et al., Editor(s): Cleland, J. L., Craik, C. S., Protein Eng. 129-161, Publisher: Wiley-Liss, New York, N.Y. (1996); WO 98/27203; Kealey et al., Proc. Natl. Acad. Sci. USA 95: 505-509 (1998); xe2x80x9cInsect Cell Culture: Recent Advances, Bioengineering Challenges And Implications In Protein Production,xe2x80x9d Palomares, et al., Editor(s): Galindo, Enrique; Ramirez, Octavio T., Adv. Bioprocess Eng. Vol. II, Invited Pap. Int. Symp., 2nd (1998) 25-52, Publisher: Kluwer, Dordrecht, Neth; xe2x80x9cBaculovirus Expression Vectors,xe2x80x9d Jarvis, Donald L., Editor(s): Miller, Lois K., Baculoviruses 389-431, Publisher: Plenum, New York, N. Y. (1997); xe2x80x9cProduction Of Heterologous Proteins Using The Baculovirus/Insect Expression System,xe2x80x9d Grittiths, et al., Methods Mol. Biol. (Totowa, N. J.) 75 (Basic Cell Culture Protocols (2nd Edition)) 427-440 (1997); and xe2x80x9cInsect Cell Expression Technology,xe2x80x9d Luckow, Verne A., Protein Eng. 183-218, Publisher: Wiley-Liss, New York, N. Y. (1996); all of which are incorporated herein by reference.
Another consideration for expression of PKS genes in heterologous hosts is the requirement of enzymes for posttranslational modification of PKS enzymes by phosphopantetheinylation before they can synthesize polyketides. However, the enzymes responsible for this modification of type I PKS enzymes, phosphopantetheinyl (P-pant) transferases are not normally present in many hosts such as E. coli. This problem can be solved by coexpression of a P-pant transferase with the PKS genes in the heterologous host, as described by Kealey et al., Proc. Natl. Acad. Sci. USA 95: 505-509 (1998), incorporated herein by reference.
Therefore, for the purposes of polyketide production, the significant criteria in the choice of host organism are its ease of manipulation, rapidity of growth (i.e. fermentation), possession or the proper molecular machinery for processes such as posttranslational modification, and its lack of susceptibility to the polyketide being overproduced. Most preferred host organisms are actinomycetes such as strains of Streptomyces. Other preferred host organisms are pseudomonads and E. coli. The above-described methods of polyketide production have significant advantages over the technology currently used in the preparation of the compounds. These advantages include the cheaper cost of production, the ability to produce greater quantities of the compounds, and the ability to produce compounds of a preferred biological enantiomer, as opposed to racemic mixtures inevitably generated by organic synthesis. Compounds produced by heterologous hosts can be used in medical (e.g. cancer treatment in the case of epothilones) as well as agricultural applications.